Receptors that mediate E. coli chemotaxis are paradigms for a large family of bacterial chemoreceptors and a larger family of prokaryotic and eukaryotic receptors. This proposal focuses on functional mechanisms in these receptors, specifically the relation of oligomeric organization to functional activity, the multiple interactions that control adaptational modification, and functional interactions among heterologous receptors. This focus is consistent with the NIGMS mission of basic research that increases understanding of life processes and lays the foundation for medical advances. Our work involves biochemical, genetic, biophysical and structural approaches plus collaborations with prominent biophysicists, biochemists and modelers. An emerging theme in biological molecular mechanisms is the importance of higher order interactions among proteins and complexes. Such interactions are central for bacterial chemotaxis. We propose to investigate the relation between receptor interactions and function by controlling in vitro the number of potentially interacting homodimers, the smallest unit of functional receptor structure. This control will be accomplished by isolating single or a small number of homodimers in Nanodiscs, water-soluble plugs of lipid bilayer surrounded by a protein annulus, a procedure we developed in a recent study. We will define the functional properties of isolated homodimers, providing a foundation for understanding functional consequences of higher order interactions. We will characterize the minimal receptor unit that activates the chemotaxis kinase, a unit our initial studies implied is 2-3 dimers. The results will define the core unit of the ternary signaling complex and identify features that require higher interactions. Given the potential of Nanodisc technology for studying chemoreceptors and other membrane proteins that form higher oligomers, we will optimize and characterize receptor-Nanodisc preparations. Receptor covalent modifications that mediate sensory adaptation are crucial for effective chemotaxis. They are controlled by multiple receptor-enzyme interactions in complex feedback loops. We will study these interactions using steady-state and transient-state kinetics, biochemical probes and mathematical plus structural modeling. A crucial interaction involves a pentapeptide connected to the receptor body by a polypeptide linker. The linker is required for effective adaptational modification. We will investigate the functional roles and postulated flexible nature of this linker by mutagenic manipulation, molecular modeling combined with experimental tests and EPR spectroscopy. We will characterize interactions among heterologous receptors using in vitro systems for adaptational assistance and for controlling the extent of influences on signaling among heterologous receptors. This research will increase our understanding of how interactions of individual molecules create complex functions. Similar phenomena, present in many life processes, are relevant to heath and disease.